Protein denatured states remain poorly understood and yet, as the starting point for protein folding, an understanding of the conformational constraints acting upon the denatured state is critical to solving the problem of how a protein folds efficiently. Misfolding diseases, such as Alzheimer's and Parkinson's diseases, are major health problems, where the causative agents are believed to be non-native and denatured states of proteins. Thus, fundamental research on denatured proteins is essential to new insight into the genesis of these disease states. This laboratory has developed a novel strategy to probe the conformational and thermodynamic properties of unfolded proteins. The propensity for forming loops of different sizes is assessed through histidine-heme loop equilibria. Single surface histidine variants have been produced in cytochrome c, allowing loop equilibria for loops of 9 to 83 amino acids to be measured under denaturing conditions. Formation of closed loops are required in the earliest stages of structure accretion when a protein folds. This system has already yielded important insights into the deviation of protein denatured states from random coil behavior. Several key properties of unfolded proteins will be probed with this system. To understand how chain stiffness and residual structure affect contact probability in a denatured protein, the dependence of loop formation on denaturant concentration will be studied by both equilibrium and kinetic methods (specific aim 1). These studies will be complemented with NMR structural data on the denatured states of selected variants. The role of topology and excluded volume in limiting the conformational space of a denatured protein will be explored (specific aim 2) with both theoretical lattice models and cytochrome c variants which contain short disulfide bridges within the denatured state histidine-heme loop. To clarify the relationship between amino acid sequence and structural preferences early in folding, fundamental studies on the effects of sequence composition on loop equilibria (specific aim 3) will be carried out, with an emphasis on how glycine and proline modulate denatured state conformational properties. In sum, the proposed experiments use a novel thermodynamic approach to probe key parameters of protein denatured states expected to be principal modulators of early events in protein folding.